Beta-(5-imino-2-pyrrolidine-carboxamido)-propamidine



' OFF Aug. 27, 1957 R. PECK ET AL 2,304,463

BETA- 5-IMINO-2-PYRROLIDINE-CARBOXAMIDO )PROPAMIDINE Filed June 27, 19563 Sheets-Sheet l NUTRIENT MEDIUM DE TROSE CASEIN HYD.

4 v MEAT EXTRACT J-SALT H20 10- v STEWHZE AGITATE 72 HRS. I I

FERMENTA'HON FILTER MvcELwm FILTERED FERMENTATlON BROTH 1 ACTIVATEDCARBON (FiLTR Am) Awusr To PH METHANOL" H O CON ENT-IO HYD OCHLORC AC DPARTiALLY spam 2 R OHRBQN CAKE Flt-TEE OFF RE$LURRY.,WITH EXAMPLEMETHANOL ACID Fri-i It FILTER FILTRATE W SPENT CARBON PH '1-8 CAKEDISTILLRT'E EvAPonA-rE I i FILTER OFF ' Drzvma YlELOS mum" \Rc-5o canon;TREATMENT EXCHANGE RESIN gx 0.25 N HYDROQHLORIC ACID i IE COLLECTFRAo'noNs INACJ'IVE OFF SALTS EVAPORATE UNTIL SALT METHANOL BEGINS TOSEPR E INAcTIVE murskm SALTS OFF REPEAT INAQTIVE FILTER SALTS i oFF OOOLE A E LET STAND 2- NlTRO PROPANE FuJRATE E. I GFF WASH ems-m LS FILTRATEf OFF I CRYSTALLINE. NOFORMICIN HYDROCHLORIDE INACTIVE COOL e-NITROPROPANE F'LT RATE FILTER CRYST'A LS FILTRRTE DRY PURE NOFORMICINHYDROCHLORIDE I NVENTORS ROBERT l PECK HENRY M. scHAFER i ATTORNEY Aug.27, 1957 R. L. PECK ET AL BETA-( 5-IMINO-2-PYRROLIDINEJ-CARBOXAMIDO)PROPAMIDINE 3 Sheets-Sheet 2 Filed June 27, 1956 030% OWE-EDA- 6mJQZtIhMZiMER O- O I Om JQZ-wIPMZ TEM mjuz xmv ZSDJOO FORM .rzwahm OUENFZK wm wm "-30: v JOOU Aug. 27, 1957 R. L. PECK ETAL 2,804,463

BETA-(5-1MINO-Z-PYRROLIDINEI-CARBOXAMIDO)-PROPAMIDINE Filed June 27,1956 s Sfieets-Sheet 3 WAVE NUMBERS m CM WAVE LENGH IN MICRQNS INVENTORSROBERT L. PECK HENRY M. SHHFER B FRANK J. WOLF ATTORNEY 23%,453 PatentedAug. 27, F357 2,804,463 BETA-(S-HVIINO-2-PYRROLlDH-iE-CARBOXAO)- PROPHNERobert Lawrence Peck, deceased, late of Plainfield, N. .l., by HelenMcL. Peck, administratrix, Binghamton, N. Y., and Henry M. Shafer,Elizabeth, and Frank Ll. Wolf, Westfield, N. J., assignors to Merck &Co., Inc, Rahway, N. J., a corporation of New Jersey Application June27, 1956, Serial No. 594,267

20 Claims. (Cl. 260-3263;)

This invention relates to a new antiviral substance and to proceduresfor producing the same from broth formed in submerged aeratedfermentation with the organism Nocardia formica. More particularly, theinvention relates to the production of an anti-viral substance bysubjecting a fermentation broth of the class described to a plurality ofadsorption, elution and chromatographic procedures yielding purifiedconcentrates having antiviral activity, and further treating suchpurified concentrates to obtain crystalline acid salts of the antiviralsubstance. This application is a continuationin-part of copendingapplication Serial No. 377,238, filed August 28,1953, now abandoned.

The new antiviral substance is a nitrogeneous, basic, water-soluble,dialysable, somewhat solvent-soluble, colorless organic substanceforming a dihydrochloride and similar acid addition salts. The structureof this substance (shown as the dihydrochloride salt) is as follows:

The structure corresponds with the name ,B-(S-imino-Z-pyrrolidine-carbox-amido)-propamidine dihydrochloride. The coined nameNoformicin has been applied to the basic substance, and under thisterminology which may be used hereinafter in the specification andclaims the hydrochloride salt is known as Noformicin hydrochloride.

The new antiviral substance, whether in the form of a purifiedconcentrate, or a crystalline salt, has been found primarily useful inthe treatment of plant virus and particularly tobacco mosaic virus, aninfection of tobacco which causes serious damage to leaf tissue. Thissubstance also exhibits antiviral activity against swine influenza whentested in mice, thus providing a practical assay procedure which ishereinafter described.

The starting material employed in producing the new antiviral substanceis a fermentation broth obtained by submerged aerated fermentation whichthe organism Nocardia formica a newly discovered organism which has beendeposited with the Northern Regional Research Laboratory, Peoria, 111.,as culture number N. R. R. L. 24170, and which can be characterized andidentified by its cultural and morphological properties as described inthe following table:

Cultural and Morphological characteristics of Nocardia formicaMorphology:

VegetativeExtensi-ve mycelial development, no fragmentation of hyphae.=Forms ghost-filaments and cytoplasmic condensations. Submerged-Straightand curved rods; Y and V forms. Rods 0.9-1.1 x 1.3-6.0 microns.Grampositive, noneacid fast.

Gelatin stab: After four days, liquefaction. Grayishwhite coloniessubmerged. No soluble pigment.

Nutrient agar slant: Fair growth. No aerial mycelium.

Nutrient broth: Flocculcnt growth.

Starch agar: Hydrolyzed. No soluble pigment.

Glucose asparagine: Fair growth. Aerial mycelium grayish-white.

Glycerol nutrient agar: Very poor growth.

Litmus milk: Peptonized. Neutral.

Nitrate agar: Nitrites produced.

Czapeka-sucrose solution: Sparse growth.

Czapeka-sucrose agar: Very poor growth.

Potato wedge: Very poor growth.

Casein: Hydrolyzed.

Dor-sets egg medium: Excellent growth, buff-colored,

convoluted, moist. No liquefaction. Medium not discolored.

Loefflers blood serum: Excellent growth, buff-colored,

- convoluted, medium 50% liquefied. Medium and exudate brown.

Paraflin: Not capable of utilizing p araifin.

Phenol: Not capable of utilizing phenol.

Reaction in inorganic medium containing carbohydrates:

0.5% glucose-acid.

0.5% glycerolacid. 0.5% lactoseacid.

0.5% maltose-acid.

0.5% sucr0se-no change.

Other characteristics:

Aerobic. Optimum temperature-28 C. Optimum pH-7.07.5

Fermentation with the organism Nocardia formica is conducted undersubmerged aerated conditions using a sterilized nutrient mediumcontaining a complex nitrogenous nutrient, a sugar, and inorganic salts,the fermentation being continued for about 72 hours at about 28 C. Asuitable nutrient medium may contain, for example, 1% dextrose, 1%casein hydrolysate, 0.3% meat extract, 0.5% NaCl, and 97.2% water. Aftercompletion of the fermentation the mycelium is removed by filtration andthe resulting filtered broth is the fermentation product whichconstitutes the starting material for the procedures of the presentinvention. This fermentation process was disclosed and claimed in anapplication of Pollard, Serial No. 372,157 filed August 3, 1953 (nowabandoned).

The process of the present invention comprises contacting fermentationbroth produced by the organism Nocardia formica with an adsorbentmaterial, such as charcoal or a cation exchange resin deriving itsexchange capacity from carboxylic groups, separating the adsorbent fromresidual liquid, eluting the adsorbed material with an aqueous acidsolution, preferably aqueous-alcoholic acid when charcoal is theadsorbent, and then neutralizing and concentrating the eluate at leastto small volume and preferably to dryness to form a crude concentratecontaining antiviral activity. This concentrate in aqueous solution isthen subjected to chromatographic purification on an adsorbent materialsuch as charcoal or a cation exchange resin, particularly of the typederiving it exchange capacity from carboxylic groups, the chromatographbeing developed and fractions at progressively lower pH being eluted bymeans of aqueous solutions of a lower aliphatic acid such as acetic orpropionic acid, or a mineral acid, such as hydrochloric or sulfuricacid. When charcoal is used in the chromatography the aqueous solutionemployed for development and elution is preferably an aqueous alcoholicsolution, suitably acidified as above described. Antiviral activityappears in eluate fractions having a pH of about 6.0 and lower. Theacidic eluate fractions are then neutralized by means of aqueous alkalisuch as aqueous sodium hydroxide, concentrated to small volume andfreeze dried to yield antiviral concentrates which can be used withoutfurther purification in the combating of plant virus such as tobaccomosaic virus.

A purified concentrate containing the hydrochloride of the antiviralsubstance can be employed for the production of crystalline salts of theantiviral substance, preferably by first preparing the crystallinehydrochloride salt. The concentrate containing the hydrochloride neednot be dried, but can be merely concentrated to small volume.Alternatively the dried concentrate can be redissolved in water forfurther processing in obtaining crystalline products. Mineral salts arefirst .removed by adding a lower aliphatic alcohol, preferably methanol,and concentrating under reduced pressure to cause crystallization ofsuch salts, which can then be filtered off. This may be repeated severaltimes until mineral salts are substantiallyeliminated. The solventsarethen removed in vacuo until a thick slurry of crude crystals isobtained, and the crystals are collected by filtration.

The crude crystalline material is dissolved in a lower aliphaticalcohol, preferably methanol, and diluted with about five volumes of2-nitropropane. Upon cooling this mixture purified crystals separate andare recovered. By recrystallization in this manner crystallinehydrochloride of the antiviral substance can be obtained having apurity, by solubility analysis, of 9 9.8:0.2%. Recrystallization canalso be effected usingother solvent mixtures such as methanol-acetoneand methanolether, This product has an equivalentweight of 268; pKa=9.4and shows characteristic infrared adsorption'as follows: broadadsorption in the 34 region, at least two bands at 5.92 1. and 6.0;/.,and a moderate band at 6.45 t. When assayed for antiviral activityagainst swine influenza in mice accordingto the procedure hereinafterdescribed the pure hydrochloride shows a survival index (S. I.) of 2.29at 2 x 0.25 mg. dose and 1.791.91 at 2 x 0.1 mg. dose.

Other crystalline salts ofthe antiviral substance can be prepared fromthe hydrochloride, as for example by adding to a warm methanolicsolution of the hydrochloride an excess of a methanol soluble salt suchas triethylamine sulfate and cooling to crystallize the sulfate salt ofthe antiviral substance.

In some instances the conversion can be made in aqueous solution. Thu ifpicric acid is added to a warmed aqueous solution of the hydrochloride,and the solution is then cooled, yellow crystals of the picrate salt ofthe antiviral substance are obtained.

In the procedure above described the early purification by charcoalchromatography may be carried out as follows: A crude concentrate, asfor example a dried product obtained after adsorption of active materialfrom fermentation broth, elution with acidic aqueous alcohol,neutralization, and a drying, is discolored in water, and this orasimilar water solution is passed through a chromatographic column ofcharcoal, preferably mixed with cellulose fiber. The column is developedwith water, aqueous alcohol, and finally with acidic aqueous alcohol. Aratio ranging from 5 parts of charcoal to 1 part of solids( in thesolution treated) to about 50 parts 01 charcoal to one part of solidshas been found satisfactory. The alcohol should be one of the loweraliphatic alcohols such as methanol, ethanol, or propanol. Theacidifying agent employed may be any non-oxidative, nonreductive mineralacid, such as hydrochloric and sulfuric acids, aswellas lower aliphaticacids, such as acetic and propionic acids. A 0.1 N solution ofhydrochloric acid is preferred.

The active material appears in the effluent when the pH of the effluentbecomes acidic. One displacement volume usually contains the bulk of theactivity. The selected fractions are then neutralized with aqueousalkali and evaporated to dryness in vacuo, or alternatively, they may beevaporated without neutralization. The fractions thus obtained arerather gummy and may be dissolved in a lower aliphatic alcohol, such asmethanol and precipitated in granular form by adding a misciblenonsolvent for the product, such as diethyl ether or diethylether-acetone mixture.

The ion-exchange resin procedure .is the presently preferred procedureand may be carried out as follows: A crude concentrate is dissolved inwater and the solution is passed through a column containing a cationexchange resin on the sodium cycle, preferably a cation exchange resinwhich derives its exchange capacity from carboxylic groups, such asDuolite CS-lOO, produced by Chemical Process Co., and Amberlite IRC-SOresin, a product of Rohm & Haas Co. The antiviral activity remains onthe column and a large portion of the impurities pass through the columnand are eliminated. The column is then eluted with an aqueous mineralacid, such as hydrochloric or sulfuric acid, or with an aqueous solutionof a lower aliphatic carboxylic acid, such as acetic or propionic acid.One normal hydrochloric acid is pre ferred. Active material appears inthe efiluent as it become acidic, particularly below pH 6.0. Thecollected fractions of efliuent are then neutralized, filtered,concentrated and freeze dried to obtain a solid concentrate, or may beused directly after neutralizing and filtering for further processing toobtain crystalline material as above described.

While ion exchange purification has been described as following initialadsorption of active material from fermentation broth on charcoal andelution with acidic aqueous alcohol, it should be understood that acation exchange resin can be employed in the manner described toinitially recover active material from fermentation broth. A first resintreatment is then followed by a second resin chromatographic treatmentto obtain the desired concentration and purification of antiviralsubstance.

The activity of the antiviral substance against swine influenza may bedetermined by employing the following assay procedure; seed material forthe swine influenza test may be prepared by infecting 12 miceintranasally with swine influenza virus (Iowa-F15). After about 3 daysthe mice are sacrificed, the lungs are removed and ground in a mortar.The ground lungs are homogenized aseptically in saline containing aphosphate buffer at pH 7. 3, the suspension being made 5% by weight. Thesuspension is centrifuged and the supernatnet liquid distributed invials, frozen in an alcohol-Dry Ice mixture and store in Dry Ice. Thisseed material is then titratedin mice using dilutions of 10- For thistitration, the mice are anaesthetized with a chloroform-ether mixture(1:2) and 0.05 ml. of diluted seed materialadrninistered. Based on theoutcome of this titration, the infecting dose is chosen to give deathswith an average survival time of 4 to 5 days.

In the routine tests, groups of six or more mice are infected with thechosen dose of swine influenza virus. The antiviral substance fortesting is administered subcutaneously 3 and 24 hours after infecting.Evidence of activity is based on the increase in survival time oftreated mice as compared to control mice. This is expressed numericallyas the quotient obtained by dividing the average survival time of thetreated mice by the average survival time of the control mice and iscalled the survival index. In a routine test where six mice are used, asurvival index of 1.31 is considered indicative of significantprolongation of life; when twelve mice are used, an index of 1.21 isfconsidered indicative of significant prolongation of The followingexamples will serve to show how procedures for preparing activeconcentrates and crystalline products in accordance with the presentinvention can be carried out, but it is tobe understoodthat theseexamples aregiven by way of illustration and not oflimitation. Example 1which showsthe preparation of the ample II is illustrated by the flowsheet of Fig. 3 of the drawing. Example III describing an alternate forpart of the procedure shown in Example V is illustrated by the flowsheet in Fig. 4 of the drawing.

Example I A nutrient medium is prepared having the followingcomposition: 7

Percent Dextrose 1 Case in hydrolysa 1 Neat extr 0.3 NaCl 0.5 H 97.2

2,000 gallons of the above medium having a pH of about 7.0 is sterilizedby heating for 15 minutes at 120 C. The inoculum is prepared as follows:The Nacardia formica microorganism i usually stored in a soil tube orunder mineral oil on an agar slant. A 250 ml. shake flask charged withthe above-described meat medium is inoculated with the organism andshaken at 20 C. until good growth resulted (24-48 hours). This flask isthen used to inoculate a 2 liter flask containing about 750 ml. of themeat medium and is shaken at 28 C. for -48 hours. This broth is thenused to inoculate a 300 gallon tank containing the same meat medium andis allowed to ferment for hours at 28 C. This 300 gallon batch is thenused as inoculum for the 2000 gallon batch. The inoculated medium isfermented under submerged condistance may be concentrated to drynessyielding a crude, solid concentrate containing the bulk of the antiviralactivity. The concentrate can be dissolved in water for assaying orfurther processing.

Example III Sixty grams of a solid concentrate prepared as described inExample II was stirred for one-half hour with 1 liter of 90% methylalcohol. The insoluble material was removed and weighed 2.5 g. (S. I.0.95 at 5-mg. dose). To the residual solution was added 2 liters ofacetone causing the precipitation of 13.5 g. of amorphous solid (S. I.1.12 at S-rng. dose). The mother liquor from this precipitation wasevaporated in vacuo to a syrup and freeze dried to give 46.3 g. of solidproduct (S. I., 1.1 at S-mg. dose). A 45.6 g. sample of the latterproduct was dissolved in water for chromatographic purification.

A column 6.5 cm. in diameter and cm. high was packed with a dry mixtureof 200 g. each of charcoal (Darco G-60) and cellulose fiber. Distilledwater was allowed to run into this column of adsorbent until onecolumnvolume had entered the adsorbent layer. At this time (after introductionof 1150 ml. of water) the solution described above (188 ml.) was runinto the column. An additional 1.5 l. of water was then used fordevelopment and was followed by 1.5 l. of 50% methanol, 1.5 l. of

70% methanol, and two liters each of 2% acetic acid and 5% aceticacid'in 70% methanol.

Eluate fractions of approximately 500 ml. each were taken. Thesefractions were evaporated to a small volume DATA ON CHROMATOGRAPHICFRACTIONS tions with agitation and forced aeration for 72 hours at s. I.28 C. The consumption of dextrose and pH of the FractionlNo 12 g fimedium is shown in the following table: fidmg. 056 Time (Hours) pHDextrosle, 45 5 550 0 14 0 95 mg'lm 6:: n 470 0: 44 1109 660 15.80 1.126.6 550 3.58 1.05 as 555 1.08 1.12 7.4 7.8 8.2 as 635 1.98 1.21 600 1.4s 0. 93 The mycelium is then removed by filtration to yield 12 thefermentation product. B

Example 11 Filtered fermentation broth obtained as described in 228 82gExample I above was slurried with 1% charcoal (Darco G-) and about 0.2%filter aid (Supercel) for a few hours. The mixture was then filtered andthe filtrate discarded. The charcoal-Supercel cake was slurried with 5901.91 1.30 2 x 100 gallons of 90% methanolic hydrogen chloride $13 11 gg(pH 2), filtered and the filter cake discarded after wash- 940 1.86 1.a0 ing. The combined filtrates were neutralized with sodium hydroxideand concentrated in vacuo to about 25 gallons at a temperature below 40C., water was added and the 475 30 30 methanol striped oif under reducedpressure to a final 740 volume of about 25 gallons. The resultingaqueous solu- Starting mate i L L12 tion of antiviral substancecontained some precipitated solids whichwere removed by filtration. Thesolution thus obtained had a total solids content of 80 mg./m1. and asurvival index of 1.44 at a 9 mg. dose level in the standard swineinfluenza virus assay on 12 mice.

Alternatively, the methanol solution of antiviral sub- 1 Fraction 5represents the first filtrate from the column; fractions 5 and 6 thusrepresent most of the water pre-wash.

2 One column-volume was 1150 ml.

Example] V A. crude solution. prepared as described in: Example :11(survival index of 1.44 at a 91mg; dose and1=17- ata 3 mg; dose orapproximately 1.34 ata mg; dose level), con: taining 7,300 grams ofsolids (total volume about 91 liters) was put through a column 4.7 cm.in diameter and 146 cm. highcontaining 1,800 grams of a carboxylic typecation exchange resin (Amberlite IRS-50; a' cation exchangeresinobtained by the copolymerization'ofmeth acrylic acid and divinyl benzenewhereintheidivinylbenzone-constitutes 2 /2 to 5% of'the'resincomposition) on the sodium cycle. The solution was allowed to flowthrough" the column at a rate of approximately 3.5 liters/ hour. Water(4 liters) was used as the-:first' developing solvent followed by 0:25 Nhydrochloric acid; The pH of the eluate-wasdeterminedperiodically andthe receiver changed when a definite drop-inth'e'pH was noticed; Theacidic fractions were neutralized with sodium hydroxide, concentratedunder reduced pressure to a small volumeand' freeze dried. The dataobtained on the individual fractionsof this experiment are shown in thefollowing table:

l The activity of the product was assayed by the routine mouse assaypreviously described.

I 0.25 N H01:

Example V The neutralized rich cuts from an IRC-SO column (similar tothose obtained in Example IV above) were concentrated in a circulatingevaporator below 40 C. until the inorganic salts began to crystallize.The concentrate was diluted with 2 volumes of methanol and cooled. Afterremoval of salts by filtration, the concentration and methanol dilutionwere repeated twice more. Finally the solution was concentrated until asyrupy solution remained.

This solution was evaporated to a thick mush of crys-' tals: in a vacuumdesiccator over CaClz. The solid crys talline material was removed byfiltration andslurried' with 3 x ml. portions of ethanol and dried.

30 g. of the material thus obtained was dissolved in 600 ml. ofmethanol. The solution was filtered through a sintered glass funnel andthe insoluble product washed with 120 ml. of methanol. The filtrate was.diluted with 3600 ml. of 2-nitropropane and chilled over night- (0-5"0.). The crystalline product was then filtered, washed with three smallportions of acetone, and dried. The crystallineproduct weighed 5.10 g. a

This sample was combined with 10 g. of material obtained in the samemanner and dissolved in about 550 ml; of methanol by warming to 50 C.The warmsolution was filtered and diluted with methanolup to a totalvolume of 600 ml. To this methanol solution was added 3,000 ml. ofZ-nitropropane whereuponthe product began to crystallize from solutionimmediately (crystallizationbegan after 2 liters hadbeenadded). Theresulting mixture was chilled (0-5" C.) over night, filtered, washed 76.53%, N=25193-%, Cl=26.25%. Found: C=36.5%-, H=6;0%-,.N=-257.8%,Cl=24.6%.

When tested for optical activity the value (cons. 1% in water) =+07.When made alkaline the specific rotation of -16 is-observed. Bothofthese specific rotations, however, are too low to be of particularsignificancec The infra-red absorption curve for the crystallinehydrochloride, mulled in petrolatum is shown in Fig. 5 of the drawing.This curve shows broad infra-red absorption in the 34,;. region, atleast two bands at 5.92 and 6.0;.t, and a moderate band at 6.45

Survival index equaled 1.91 at 2 x 0.1 meg; dose ('12 mice,subcutaneous).

It will. be. evident from the structural formula that Noformicin and.its salts. can exist in two isomeric forms,v audit hasbeen foundthatonly. the isomer derived from fermentation broth. is active. This isevidenced by the fact'that the synthetically prepared hydrochloride,which is a racernic mixture shows only one-half the activity when testedagainst swine influenza in mice in accordance with the assay procedureherein described.

Example VI The hydrochloride salt (0.25 gm.) prepared as described inExample V was dissolved in 10 ml. of warm methanol and a methanolicsolution (3.4 ml.) containing 0:46 gm. of triethylamine sulfate wasadded to the solution; of sulfate; Material began to crystallize beforeall the solution of sulfate was added. Crystallization was broughtto-complction by cooling one hour at 010 C.

The crystalline precipitate was centrifuged off, washed with ether anddried. Itwas then dissolved in about 1 ml. of water and the solution wasdiluted dropwise with anequal volume of methanol (crystallization beganbe fore all the methanol was added). The crystalline sulfate wasseparated by centrifugation, washed twice with methanol and dried, wt.0.29 gum. and melted with decomposition at about 263 C.

Partial analysis.--Found: C, 33.69; H, 5.88; N, 23.3; S04, 32.6%.

The sulfate and hydrochloride salts of the antiviral substance show nocharacteristic ultraviolet peaks andvery little absorption over therange 2300 to 4000 A.

Example VII Example VIII Approximately pounds (2 cu. ft.) of AmberliteIRC-SO resin onthe sodium cycle was positioned in a column 6' inches indiameter and 11 feet long. The resin was flushedwith several'volurnes ofwater. 1000 gallons of a filteredfermentation broth prepared asdescribed in Example I was passed through the column at the rate ofabout 50 gallons per hour. The column was washed with water and theactivity selectively eluted with 0.1 N hydrochloric acid at a rate ofabout 50 gallons per hour. The acidic eluate was periodicallyneutralized with sodium hydroxide. The various eluates thus obtainedwere concentrated under reduced pressure to a final volume' of about 1gallon. (IO-20% total solids) and then This is about 1.5 times thetheoretical amount freeze dried. The solids thus obtained were assayedby the standard swine influenza virus assay using 12 mice.

The following table demonstrates typical results obtained by thisprocess:

A 51 g. sample of antiviral concentrate obtained as described in ExampleIV (survival index 1.66 at 2 x 3 mg. dose, 1.08 at 2 x 1 mg. dose) wasdissolved in one liter of water. The solution was adjusted to pH 6.5 andfiltered to remove a trace of insoluble material. The clear, darkfiltrate was used for the rechromatographic experiment (an aliquotshowed survival index 1.43 at 2 x 3 mg. dose).

A column was prepared with IRC-SO resin on the sodium cycle to give alayer of resin x 35" containing 250 ml. of the moist resin. The columnwas washed with water and was then used. 7

The filtrate containing the antiviral substance was allowed to percolatethrough the column at a rate of 34 ml. per minute. Development wascarried out with one liter of water, 78 liters of dilute aqueoushydrochloric at pH 2.5, 3.2 liters of dilute hydrochloric acid at pH1.1, and 3.6 liters of 0.2 N hydrochloric acid. Efiluent fractions werecollected as shown in the accompanying table at appropriate pH changes.Each fraction was concentrated to small volume and freeze-dried. Weightsof fractions and assay values are shown in the table.

RECHROMATOGRAPHY ON IRC50 RESIN Survival Index Vol. Wt. of FractionLiter pH of Eluate solids,

g. 2x3n1g. 2x1.5mg.

dose dose 1 7. 39. 4 1. 38 9 7.0 to 5.7 9. 6 1. 51 7 5. 7 5. 8 1. 30 75. 7 2. 9 1. 56 20 5. 7 4. 3 1. 64 20 5.7 to 5.0 5. 8 1. 77 4 5.0 to 4.90.45 2.16 X 4 4.9 to 4.7 1. 49 2.05 0.55 4.7 to 3.8 0.12 3. 4 3.8 to2.9 1. 24 1. 90 2. 4 2.9 to 2.6 O. 55 1. 8 2.6 to 1.2 0. 44 1. 35 1.2 to1.1 0. 06 3.6 below 1 0.01

X. One dose only; sample rather toxic. XX. This value obtained with 2 x0.25 mg. dose.

Example X To 150 gallons of broth (about pH 8.0) prepared as describedin Example I was added 1% I-Iyflo Supercel with agitation. The broth wasfiltered through a filter press followed by 10 gallons of water-wash.One percent Darco G-60 was added to the filtered broth and stirred /2hour after which time 0.5%, Hyflo Supercel' was added. This slurry wasfiltered through a filter press and the cake exhausted for /2 hour,washed with 10 gallons of water and exhausted for one hour. The dampcake was removed from the press and extracted twice with $4 the originalbroth volume of 85% aqueous methanol adjusted to pH 2.0 withhydrochloric acid. The rich methanol extracts were kept separate andneutralized to pH 7.0 with 30% sodium hydroxide, concentrated at 40 C.to 20% solids and freeze dried. Eluate I weighed 95 grams; S. I. 1.51(2x15 mg. dose). Eluate II weighed 56 grams; S. I. 1.31 (2x15 mg. dose).

Example XI An antiviral substance obtained as described in Example IIIhaving a survival index of 1.97 at 5. mg. was tested for elfect on themultiplication of tobacco mosaic virus in tobacco leaf tissue. Turkishtobacco plants infected with tobacco mosaic virus were employed for thestudy. One set of infected plants served as the control for theexperiment, a second set of infected plants was treated with a 0.1%solution of the antiviral agent. The antiviral agent caused no visibleinjury to the leaf tissue.

h The virus content of the control set and the treated set terminationof the optical density of the virus suspension.

The quantity of tobacco mosaic virus contained in the treated plants wasdecreased 69%, as compared with the quantity of virus contained in thecontrol plants. The test has been repeated several times and the degreeof decrease of virus multiplication has ranged from 70 to This decreasein virus content of an economically valuable plant is highlysignificant.

The term antiviral substance as applied herein to Noformicin compoundsis to be understood as separate and distinct from the term antibioticsubstance as the latter term is normally used.

Noformicin hydrochloride has no significant antibiotic activity as willbe evident from the following tabulation showing comparative activitiesof the common antibiotics and Noformicin hydrochloride against 12representative organisms.

Organisms Material tested Amount in(mcg.)

A B o D E F G H I .T K L 200 12 21 16 '20 30 2s 23 27 Terramycm 50 9 16.s 14 g1 12; g: 200 16 22 16 23 1 Chmmmycetm 50 1s 17 10 14 1s 16 10 17125 PemcmmG 12 X 27 1s 2s 3 a 12 a I 200 13 11 13 1 Neomycm 50 9 s 10'1a 8 11 4 s a a a a -15 2 a Bacitracin-.- 50

i 12 X X X X 14 X 12 Streptomycin 7 6 7 11 1g Notormicin-hydrochloride 611 5 8 19 ""l l' 15 X=Not tested.

number recorded is that of the diameter of' the zone; of. inhibition ofthe test organism aroundl the cup contammg.

the sample. The first seven organisms a-reusually con.-

sidered gram negative organisms whereas the last fiveare gram positiveorganisms. It is readily-apparentv that even at a concentration 10,000meg. whichis 20 to 3333 times that of the above antibiotics thatNoformicin exerts only a small effect on a few of the organisms whereasall of the other antibiotics are much more active. Although it isdifficult to estimate the order of activity'precisely it is consideredthat the abovedata indicate that Noform-icin has about 1% as muchactivity as an ordinary antibiotic. Since the common dosages of theseantibiotics is about l-2 g. per day per. man, it is readily apparentthat Noformicin, which would require a dosage of 1.00- 200 g. per day toexert similar activity, is not anantibiotic in the usual sense of theword.

Various changes and modifications in the procedures herein described mayoccur to those skilled in the art, and to the extent that such changesand modifications are embraced by the appended claims, it istobeunderstood that they constitute part of our invention.

We claim:

1. The process that comprises contacting fermentation broth produced bythe organism Nocardia formica with an adsorbent material selected fromthe group consisting of charcoal and cation exchange resins, eluting theadsorbed material with an aqueous acidic solution which also containsalcohol when charcoal is the adsorbent, transferring the eluate to asecond adsorbent material arranged in a chromatographic column andselected from the group consisting of charcoal and cation exchangeresins, developing the column with water which also contains alcoholwhen the adsorbent is charcoal, eluting the column with aqueous acidsolution which also contains alcohol when the adsorbent is charcoal, andcollecting fractions of eluate having a pH below about 6.0 andconcentrating the collected eluate to thereby obtain a substance havingantiviral activity.

2. The process as defined in claim 1 wherein the acid employed in atleast one of the elution steps is a mineral acid.

3. The process as defined in claim 1 wherein the acid employed in atleast one of the elution steps is a lower aliphatic carboxylic acid.

4. The process as defined in claim 1 wherein the adsorbent materialemployed in at least one of the adsorption steps is charcoal, and anaqueous-alcoholic acid solution is employed for eluting adsorbedmaterial from the charcoal.

5. The process as defined in claim 1 wherein the-ad'- sorbent materialemployed in at least one of the adsorption steps is a cation exchangeresin deriving its exchange capacity from carboxylic groups.

6. The process as defined in claim 1 wherein the acidic eluate isneutralized prior to concentration.

7. The process as defined in claim 1 wherein the collected eluate isconcentrated to small volume and freeze dried to obtain a crudeantiviral substance in solid form.

8. The process as defined in claim 1 wherein the eluate fractions areobtained by eluting with a hydrochloric acid solution and are furthertreated by neutralizing the excess acid therein, concentrating to smallvolume, adding methanol to crystallize out inorganic salts,concentrating the residual solution to precipitate crude antiviralhydrochloride, and purifying the same by recrystallizing from a solventmixture comprising about 1 part of methanol to 5 parts of2-nitropropane.

9. The process for recovering an antiviral substance from a fermentationbroth produced by the organism Nocardia formic-a that comprisescontacting the fermentation broth with an adsorbent selected from thegroup consisting of charcoal and cation exchange resin, removing theresidual broth, eluting adsorbed material with an aqueous acidicsolution which also contains alcohol when the adsorbent is charcoal, andconcentrating the resulting eluate to small volume.

10. The process for recovering an antiviral substance from afermentation broth produced by the organism Nocardia formica thatcomprises contacting the fermentation broth with charcoal, removing theresidual broth, eluting adsorbed material from the charcoal with anaqueous-alcoholic acidic solution, and concentrating the resultingeluate to small volume.

11. The process for recovering an antiviral substance from afermentation broth produced by the organism Nocardia formica thatcomprises contacting the fermentation broth with a cation exchange resinderiving its exchange capacity from carboxylic groups, removing theresidual broth, eluting adsorbed material from the resin with an aqueousacidic solution, and concentrating the resulting eluate to small volume.

12. The process for purifying an antiviral substance derived from afermentation broth produced by the organism Nocardia formica thatcomprises adding an aqueous solution of crude antiviral substance to 'achromatographic column of adsorbent material, developing and eluting thecolumn with an aqueous acidic solution, and collecting and concentratingfractions of eluate having a pH below about 6.0.

13. The process as defined in claim 12 wherein the adsorbent material isa cation exchange resin deriving its exchange capacity from carboxylicgroups.

14. The process as defined in claim 12 wherein the adsorbent material ischarcoal and the eluting solvent is an aqueous-alcoholic acidicsolution.

15. The process for obtaining in crystalline form the antiviralsubstance derived from a fermentation broth produced by the organismNocardia formica that comprises providing a concentrated aqueoussolution of crude hydrochloride salt of the antiviral substance,crystallizing out inorganic salts by addition of methanol, concentratingthe residual solution to precipiate the hydrochloride, re-

dissolving the precipitate in methanol, adding about 5 volumes of2-nitropropane, and chilling the resulting mixture to crystallize thehydrochloride salt of the antiviral substance.

16. An antiviral substance selected from the group consisting of acidsalts of fl-(5-imino-2-pyrrolidinecarboxamido) -propamidine.

17. An antiviral substance as defined in claim 16 in the form of thehydrochloride salt.

18. An antiviral substance as defined in claim 16 in the form of thesulfate salt.

19. An antiviral substance as defined in claim 16 in the form of thepicrate salt.

20. The crystalline antiviral substance of the formula No referencescited.

16.AN ANTIVIRAL SUBSTANCE SELECTED FROM THE GROUP CONSISTING OF ACIDSALTS OF B-(5-IMINO-2-PYRROLIDINECARBOXAMIDO)-PROPAMIDINE.